The chromogenic assay used to monitor the potency of factor VIII does not consistently give the same result as a plasma substrate-based test. The standard measurement of factor VIII coagulant activity at the FDA employs the plasma substrate assay, while the European Pharmacopoeia mandates a chromogenic test. Potency discrepancies between these two tests affect product fill and patient care. Furthermore, the replacement of plasma- derived products with recombinant equivalents has led to additional concerns about the comparability of these products and the applicability of current assay systems. Therefore, new testing procedures and/or molecular-based testing methods need to be developed in order to reliably assay both plasma-derived and recombinant-technology products. To understand the fundamental mechanisms and resolve the disparities between the tests and to develop assay methods for these products, we are evaluating molecular parameters that influence the assays and are attempting to develop analytical methods based on surface plasmon resonance (SPR) biosensor technology measured using BIA core%. Strategy 1: Examine the activation kinetics of three recombinant-derived factor VIII products during thrombin proteolysis to determine whether B-domain deletion plays a role in thrombin susceptibility. Strategy 2: Study the interaction kinetics and catalytic activities of rFVIII products when complexed with factor Ixa on a supported hybrid bilayer membrane (HBM) to determine the effect of phospholipids on the coagulation process. HBM is a user-defined artificial phospholipid monolayer generated on an HPA sensor chip. The HBM will provide a native environment to membrane-bound coagulation molecules. Finding 1: The proteolysis of three rFVIII products was examined, two of which contained the B-domain and the third of which did not. Our results showed that rFVIII product without the B-domain was cleaved and dissociated faster from von Willebrand factor (vWF) by thrombin than products with the B-domain. Our data suggest that the B- domain affects the interaction of FVIII with vWF, and modulates the susceptibility of FVIII to thrombin cleavage. Finding 2: Unilamellar vesicles were generated by reverse-phase evaporation of 1 -plamitoyl-2- oleoyl-sn-glycero-3-phosphocholine (PC) and 1 -palmitoyl-2-oleoyl-sn- glycero-3-[phospho-L-serine] (PS) at a ratio of 80:20. Using this liposome preparation, HBM was stably formed on the HPA sensor chip. Our preliminary data indicated that the molecular complex of rFVIIIa and FIXa was generated on the HBM. We are currently investigating the binding kinetics and the enzymatic activities of different rFVIII products when in complex with membrane-bound FIXa.